Inhibition of mTOR via an AAV-Delivered shRNA Tested in a Rat OIR Model as a Potential Antiangiogenic Gene Therapy

Abstract

Citation: Lee SHS, Chang H, Kim JH, et al. Inhibition of mTOR via an AAV-Delivered shRNA tested in a rat OIR model as a potential antiangiogenic gene therapy. Invest Ophthalmol Vis Sci. 2020;61(2):45. https://doi.org/10.1167/iovs.61.2.45 PURPOSE.Recent studies have shown that inhibitors of the mechanistic target of rapamycin (mTOR) play important roles in proliferating endothelial cells within the retinal vasculature. Here we explore the effects of inhibiting mTOR as a potential gene therapeutic against pathological retinal angiogenesis in a rat model of oxygen-induced retinopathy (OIR). METHODS.Sprague-Dawley pups were used to generate the OIR model, with a recombinant adeno-associated virus expressing an shRNA (rAAV2-shmTOR-GFP) being administered via intravitreal injection on returning the rats to normoxia, with appropriate controls. Immunohistochemistry and TUNEL assays, as well as fluorescein angiography, were performed on transverse retinal sections and flat mounts, respectively, to determine the in vivo effects of mTOR inhibition. RESULTS.Compared with normal control rats, as well as OIR model animals that were either untreated (20.95 ± 6.85), mock-treated (14.50 ± 2.47), or injected with a control short hairpin RNA (shRNA)-containing virus vector (16.64 ± 4.92), rAAV2-shmTOR-GFP (4.28 ± 2.86, P = 0.00103) treatment resulted in dramatically reduced neovascularization as a percentage of total retinal area. These results mirrored quantifications of retinal avascular area and vessel tortuosity, with rAAV2-shmTOR-GFP exhibiting significantly greater therapeutic efficacy than the other treatments. The virus vector was additionally shown to reduce inflammatory cell infiltration into retinal tissue and possess antiapoptotic properties, both these processes having been implicated in the pathophysiology of angiogenic retinal disorders. CONCLUSIONS.Taken together, these results demonstrate the strong promise of rAAV2-shmTOR-GFP as an effective and convenient gene therapy for the treatment of neovascular retinal diseases.

AAV expressing an mTOR‐inhibiting siRNA exhibits therapeutic potential in retinal vascular disorders by preserving endothelial integrity

“…support our previous findings that suggested an effective reduction of neovascularization by AAV2-shmTOR in CNV and OIR models [8,20]. Retinal vascular diseases share salient and common features of retinal neovascularization.…”

“…In our previous studies, we demonstrated that inhibition of the mTOR signaling pathway using a small‐hairpin RNA (shRNA) delivered via recombinant adeno‐associated virus 2 (AAV2) (termed rAAV2‐shmTOR‐SD, renamed here as AAV2‐shmTOR) led to an effective reduction of neovascularization in the mouse choroidal neovascularization (CNV) and rat oxygen‐induced retinopathy (OIR) models [ 18 , 19 , 20 ]. In those results, we confirmed the successful transduction of AAV pseudotyped with serotype AAV2 on the retinal tissues.…”

Viral-Vector-Delivered Anti-Angiogenic Therapies to the Eye

“…In a rat model of hVEGF, suprachoroidal injection of RGX-314 between the sclera and choroid suppressed VEGF-induced vascular leakage and vasodilation [27]. Oxygen-induced retinopathy in rat, laser induced CNV in mouse AAV2 intravitreal mTOR shRNA, wet AMD, ROP, DR [75][76][77] Laser induced CNV in mouse scAAV2 subretinal endostatin wet AMD [78] Oxygen-induced retinopathy in rat, laser induced CNV in mouse scAAV2 intravitreal, subretinal calreticulin anti-angiogenic domain (CAD) wet AMD, DR [79] Oxygen-induced retinopathy in mouse rAAV intravitreal Kringle1 domain of hepatocyte growth factor (HGFK1) wet AMD, DR [80] Oxygen-induced retinopathy in mouse Adenovirus GV120 intravitreal Galetin-1-RNAinterference ROP [81] Laser induced CNV and oxygen-induced retinopathy in mouse, AAV1-CRISPR/Cas9 intravitreal CRISPR/Cas9 targeted to VEGFR 2 wet AMD, ROP [82] Laser induced CNV in mouse AAV9-CRISPR-LbCpf1 intravitreal CRISPR-LbCpf1 targeted to VEGF A and HIF1α wet AMD [83] Laser induced CNV in mouse Lentivirus subretinal microRNAs targeting VEGF-A wet AMD [62] Laser induced CNV in mouse AAV5 subretinal PEDF, multiple miRNAs targeting the VEGFA gene wet AMD [84] Corneal chemical burn in a rabbit AAV8/9 chimeric capsid (8G9) corneal intrastromal codon optimized human leucocyte antigen G1+ G5 Corneal neovascularization [85] Oxygen-induced retinopathy in rat Lentivirus subretinal shRNAs to VEGFR2 or STAT3 ROP [86] Oxygen-induced retinopathy in rat Lentivirus subretinal VEGF-A or VEGF-A164 shRNA ROP [87] AAV = adeno-associated virus, ADV = adenovirus, AMD = age-related macular degeneration, CNV = choroidal neovascularization, DR = diabetic retinopathy, LV = lentivirus, ROP = retinopathy of prematurity.…”

“…Recombinant AAV derived from self-complementary AAV2, expressing a short hairpin RNA, blocks the activity of mTOR complexes 1 and −2. Intravitreal injection of AAV-mTOR shRNA was shown to suppress angiogenesis and inflammation in a laser-induced CNV mouse model [ 75 ] and reduce neovascularization in a rat oxygen-induced retinopathy (OIR) model [ 76 ]. In another study, the optimal viral genome size for efficient AAV assembly was ensured by using a stuffer DNA derived from the 3’ UTR of the human UBE3A gene [ 77 ].…”

Viral-Vector Delivered anti-Angiogenic Therapies to the Eye

“…Recombinant AAV derived from self-complementary AAV2, expressing a short hairpin RNA blocks the activity of mTOR complexes 1 and -2. Intravitreal injection of AAV-mTOR shRNA was shown to suppress angiogenesis and inflammation in laser induced CNV mouse model [52] and reduce neovascularization in rat oxygen-induced retinopathy (OIR) model [53]. In other study optimal viral genome size for efficient AAV assembly was ensured by using a stuffer DNA derived from 3` UTR of the human UBE3A gene [54].…”